The invention relates to a method for the specific detection and identification of retroviral nucleic acids/retroviruses in an arbitrary specimen and a diagnosis kit for implementing this method. It also relates to retrovirus-specific probes for reverse dot blot hybridisation and retrovirus-specific oligonucleotide primer mixtures (MOP) comprising forward and reverse primers for producing amplificates of retrovirus-specific nucleic acids from the specimen.
Exogenous and endogenous retroviruses (HERV) are etiological agents for a multiplicity of tumorigenic diseases in humans and animals. They are involved in the formation of tumours and leukaemias in numerous animal models but also in humans (HTLV-I and II). Others also cause immunodeficiency diseases (HIV). Present research indicates that retroviruses may also play a role as triggers of autoimmune diseases (Kalden and Herrmann, 1993) and neuronal degenerative diseases such as multiple sclerosis (Tuke et al. 1997). Intensive research in the field of endogenous and exogenous retroviruses has so far led to the continual discovery of new retroviral sequences in the human genotype whose expression could possibly be associated with specific diseases. For example, the expression of Gag proteins of the HERV-K family is associated with almost all forms of testicular and ovarian germinomas (Sauter et al. 1995). Antibodies against HERV-K Env protein were detected in human sera (Vogetseder et al. 1993). The HERV-K-IDDM env gene which was isolated from patients with type-1 diabetes possibly codes for an endogenous superantigen (Conrad et al. 1997).
Statistically reliable studies using large groups of patients are required to correlate specific diseases with the activity of specific endogenous or exogenous retroviruses. The expenditure in time and money required for this using the known methods of detection according to the prior art is immense.
The increasing use of retroviral vector systems in human gene therapy casts doubts on the safety from undesirable side effects (genome changes in the target cells, transmission of undesirable viruses). Thus, a certain percentage of undesirable gene sections of endogenous or foreign retroviruses are also co-packaged in the retroviral particles for therapeutic application in packaging cell lines (Co-packaging, Sherwin et al. 1987, Scolnick et al. 1979, Takeuchi et al. 1992). For example, transcripts of certain endogenous retroviruses, such as those present in related form in the genome of packaging cell lines, have been detected in retrovirus-like particles (pseudotypes) of the breast cancer cell line T47D (Seifarth et al. 1995, 1998). The packaging of such undesirable retroviral sequences can lead to the recombination and formation of new retroviruses having modified, possible pathogenic properties. The re-integration of such recombinant retroviruses in the genome of the target cells can lead to insertion mutagenesis and consequently to inactivation of important genes of the cell cycle and possibly to tumorgenesis.
For this reason it is necessary to conduct quality control of the gene vector preparations to be used for gene therapy using a sensitive test system. This could prevent any undesirable retroviral sequences from being transfused. In the event of positive detection, a vector preparation could be subjected to suitable purification (purging) before being administered to the patient. The methods known according to the prior art are not suitable for such an application.
A controversial issue at the present time is the use of animal organs for transplantation in humans (xenotransplantation). For example, as a result of the shortage of suitable donors, heart valves from pigs are being increasingly transplanted in humans. The transplantation of heart, liver and kidney transplants is also planned. However, recent research has shown that within the transplantation framework and the associated immunosuppression by medication, endogenous or exogenous retroviruses so far suppressed in the donor organ can become activated in the recipient. As has already been demonstrated experimentally, these retroviruses of animal origin are pathogenic for certain human cell types (xenotropism) and could thus lead to a serious systemic disease of the organ recipient. In cases where pathogenic infectious virus particles are formed, transmission to uninvolved third parties (epidemic) cannot be excluded. Last but not least, recombinations of retroviruses of animal origin with endogenous human retroviruses could result in new pathogenic virus recombinants with completely new host tropisms.
There is thus a need for fast, reliable and at the same time favourably priced detection systems which could be used to test the transplant carrier for infection with retroviruses of animal origin on a regular basis.
A number of methods for direct and indirect virus detection are available for the detection of viral infections in the prior art. For the direct detection of virus particles, products of viral replication (viral antigens) or an immune response directed against the virus (antiviral antibodies) these include electron microscopy (EM), staining of viral proteins with fluorescent antibodies, “enzyme-linked immunosorbent assay” (ELISA) and radioimmunoassay (RIA). Molecular biological methods such as nucleic acid hybridisations with virus-specific gene probes (dot-blot, southern-blot, northern-blot) and polymerase chain reaction (PCR) with virus-specific primers are being conducted increasingly to directly detect the virus and its nucleic acids.
In the indirect methods it is usually not the viruses themselves but their after-effects that are detected, i.e. the changes (cytopathic effects) in cells induced by a virus replication. This must usually be conducted in an in vitro cell culture system tailored to the virus to be detected. This requires living cells in which the virus to be detected can replicate. Depending on the type of virus, cell cultures, organ cultures, embryonated chick eggs or even laboratory animals are required for the detection. The manifestation of the cytopathic effect (cell lysis, focal or diffuse cell growth, syncytium formation, rounding) and the host spectrum of the virus are used as indices to identify the virus. Frequently however, a precise identification can only be made in combination with serological or molecular biological methods (PCR).
The relatively low sensitivity of some direct methods of detection (EM, antibody staining) means that the specimen must contain a certain quantity of virus for a successful detection or it must be enriched by suitable methods (ultracentrifuging). If this is not practicable, the virus must be preliminarily cultured in a special in vitro cell culture system. Since many viruses possess special host cell tropisms, a special test system is required for each virus to be tested. This results in high laboratory costs, their evaluation is very time-consuming in some cases and requires very great experience.
Serological methods (ELISA, RIA) are generally highly sensitive and have developed into the current gold standard in virus diagnosis. However, the disadvantage of all serological methods is that a specific antibody is required for each virus to be tested. In one test run the sample to be studied can thus only be tested for one putative virus. Studies of entire expression patterns using these methods can only be made at great expense in time and cost.
Developments in the field of molecular biology have led to the development of new methods of detection (hybridisations, PCR) which possess similar sensitivity to serological antigen methods of detection. In this case also, the detection success stands or falls by the availability of virus-specific gene probes (hybridisation) or oligonucleotides (PCR). Since the use of several probes or PCR primers is limited because of non-specific interactions in a reaction formulation, many experiments must be conducted in parallel to detect complex expression patterns.
In view of the circumstances described previously, the problem for the present invention was to provide an efficient and reliable, and at the same time fast method for the multiple detection of endogenous and exogenous retroviruses of human and animal origin.